ORIGINAL ARTICLE




Short-Sequence Oligopeptides with Inhibitory
Activity against Mushroom and Human Tyrosinase
Anan Abu ...
A Abu Ubeid et al.
    Oligopeptide Tyrosinase Inhibitors




    inhibition, previous studies suggested that HQ exerts it...
A Abu Ubeid et al.
                                                                                                       ...
A Abu Ubeid et al.
    Oligopeptide Tyrosinase Inhibitors




                                                      L-Dopa...
A Abu Ubeid et al.
                                                                                                       ...
A Abu Ubeid et al.
    Oligopeptide Tyrosinase Inhibitors




    and another site specific for dopa oxidase activity. Our...
A Abu Ubeid et al.
                                                                                                       ...
A Abu Ubeid et al.
    Oligopeptide Tyrosinase Inhibitors




    Katayama K, Armendariz-Borunda J, Raghow R, Kang AH, Sey...
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Short Sequence Oligopeptides With Inhibitory Activity Against Mushroom And Human Tyrosinase

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Short Sequence Oligopeptides With Inhibitory Activity Against Mushroom And Human Tyrosinase

  1. 1. ORIGINAL ARTICLE Short-Sequence Oligopeptides with Inhibitory Activity against Mushroom and Human Tyrosinase Anan Abu Ubeid1,2, Longmei Zhao1,2, Ying Wang1,2 and Basil M. Hantash1 Cutaneous hyperpigmentation is a common disorder due to excess melanin production by the enzyme tyrosinase. The gold standard for treatment is hydroquinone (HQ), which reduces pigmentation through its toxicity to melanocytes rather than via tyrosinase inhibition. We screened an internal library for oligopeptides that inhibited both mushroom and human tyrosinase but showed no cytotoxicity to human melanocytes. We identified two highly active inhibitory sequences, P3 and P4, of 8- and 10-amino-acid-length, respectively. Mushroom tyrosinase inhibition was dose-dependent with IC50 (half-maximal inhibitory concentration) values of 123 and 40 mM, respectively, compared with 680 mM for HQ. Other oligopeptides showed weaker or no inhibitory activity. Kinetic studies showed that P3 and P4 are competitive inhibitors of mushroom tyrosinase. At 100 mM, P3 and P4 inhibited human tyrosinase by 25–35%. This inhibition partially depended on whether L-dopa or L-tyrosine was the substrate, suggesting that tyrosinase may contain contains two distinct catalytic sites. Treatment of melanocytes with 100 mM P3 or P4 for 7 days led to a 27 or 43% reduction in melanin content. This inhibition was independent of cell proliferation and cytotoxic effects. Our data suggest that peptide-mediated inhibition of melanogenesis is due to reduction in tyrosinase activity. Journal of Investigative Dermatology advance online publication, 14 May 2009; doi:10.1038/jid.2009.124 INTRODUCTION contributing to lower productivity, social functioning, and Melanin, the end product of melanogenesis, determines the self-esteem (Finlay, 1997). color of human skin, hair, and eyes and is synthesized within Melasma is an acquired pigmentary disorder that occurs in unique membrane-bound organelles called melanosomes. women of all racial and ethnic groups (Rendon et al., 2006). The type and amount of melanin and its distribution pattern Although the cause of melasma is unknown, factors include in the surrounding keratinocytes determine the actual color of genetic predisposition, UV light exposure, and estrogen skin (Parvez et al., 2006). Melanogenesis is regulated by (Johnston et al., 1998). PIH represents a pathophysiological various environmental, hormonal, and genetic factors, such response to cutaneous inflammation, such as acne, atopic as UV exposure, a-melanocyte-stimulating hormone, mela- dermatitis, lichen planus, chronic dermatitis, and phototoxic nocortin 1 receptor, and agouti-related protein (Kadekaro eruptions (allergic reactions to drugs coupled with exposure et al., 2003; Petit and Pierard, 2003). The functions of to sunlight) (Lacz et al., 2004). Tyrosinase is a multifunctional melanin include protection of skin from UV radiation, the copper-containing oxidase, and is considered the key inhibition of photocarcinogenesis, and the synthesis of enzyme orchestrating mammalian melanogenesis (Sanchez- vitamin D3 (Lindquist, 1973). Hyperpigmentation is a very Ferrer et al., 1995). This key enzyme catalyzes two distinct common and usually harmless disorder in which patches of reactions in the biosynthesis pathway of melanin: the skin become darker in color than the normal surrounding hydroxylation of a monophenol (tyrosine) and the conversion skin. This darkening occurs when an excess of melanin is of an o-diphenol (dopa) to the corresponding o-quinone formed and deposited in the skin. The most common (dopaquinone). A multitude of different tyrosinase inhibitors pigmentation disorders for which patients seek treatment are used today to treat epidermal hyperpigmentation condi- are melasma and post-inflammatory hyperpigmentation (PIH) tions. Skin-whitening agents in the cosmetics industry include (Lynde et al., 2006). These conditions may have a major hydroquinone (Jimbow et al., 1974), arbutin (Maeda and impact on a person’s psychological and social well-being, Fukuda, 1996), kojic acid (Cabanes et al., 1994), vitamin C (Kojima et al., 1995), retinol (Pathak et al., 1986), azelaic 1 acid (Schallreuter and Wood, 1990), and other botanicals. Elixir Institute of Regenerative Medicine, San Jose, California, USA 2 The most popular depigmenting agent, hydroquinone These authors contributed equally to this work (dihydroxybenzene; HQ), has been in use since 1961. HQ, Correspondence: Dr Basil M. Hantash, Elixir Institute of Regenerative Medicine, 5941 Optical Court, San Jose, California 95138, USA. with formulations of 2–4%, has been widely used for E-mail: basil@elixirinstitute.org melasma treatment and reports of its clinical efficacy have Abbreviations: [E], enzyme concentration; HQ, hydroquinone; [I], inhibitor; been well documented (Ennes et al., 2000). HQ inhibits the PIH, post-inflammatory hyperpigmentation; [S], substrate concentration conversion of dopa to melanin by inhibiting the activity of Received 16 February 2009; accepted 1 April 2009 tyrosinase (Palumbo et al., 1991). In addition to tyrosinase & 2009 The Society for Investigative Dermatology www.jidonline.org 1
  2. 2. A Abu Ubeid et al. Oligopeptide Tyrosinase Inhibitors inhibition, previous studies suggested that HQ exerts its effect attention to short-sequence peptides as potential therapeutic on melanogenesis through degradation of melanosomes and candidates for the treatment of skin disorders. The most destruction of melanocytes. It was also found that alteration widely studied and popular signal peptide is the sequence of melanosome function, depletion of glutathione, generation lysine–threonine–threonine–lysine–serine (KTTKS) found in of reactive oxygen species, and subsequent oxidative damage type-I procollagen (Katayama et al., 1993). This pentapeptide of membrane lipids and proteins may play a role in the has been shown to stimulate feedback regulation of new depigmenting effect of HQ (Briganti et al., 2003). HQ is collagen synthesis and to result in an increased production of considered to be cytotoxic to melanocytes, potentially extracellular matrix proteins such as types I and II collagen mutagenic to mammalian cells (Curto et al., 1999), and and fibronectin, significantly improving the appearance of causes skin irritation (Parvez et al., 2006). Owing to the long- photoaged human facial skin (Robinson et al., 2005; Lupo term risk of mutagenesis (DeCaprio, 1999), the use of HQ in and Cole, 2007). cosmetics has been banned by the European Committee (24th In this study, we screened an internal oligopeptide library Dir. 2000/6/EC) and formulations of 4% are available only by for inhibitory activity against tyrosinase and the absence of prescription in selected other countries (Solano et al., 2006). melanocyte toxicity. We discovered several short-sequence This has led to the use of alternative agents such as kojic peptides that met both these criteria. acid and arbutin. However, these agents failed to show efficacy in vivo, as clinical trials have been disappointing to RESULTS date (Curto et al., 1999). The efficacy of these products Inhibition of mushroom tyrosinase by oligopeptides appears to be limited due to their adverse effects, poor As the rate-limiting enzyme in melanin production, tyrosi- skin penetration, and low formulation stability (Hermanns nase was targeted during the screening of seven potential et al., 2000). More recently, investigators have turned their hypopigmenting agents (Table 1). Of these oligopeptides, two showed substantial inhibitory effects on mushroom and human tyrosinase activity, whereas the other peptides had Table 1. Oligopeptide inhibitory profile against either poor or no inhibitory activity on tyrosinase. Dose– mushroom tyrosinase response curves were obtained after an incubation time of Oligopeptide Sequence IC50 [M]1 120 minutes (Figure 1). Peptide P6 had no effect on tyrosinase activity, whereas peptides P1, P2, P5, and P7 showed an P1 SFLLRN 8 mM inhibitory effect at high concentrations. On the other hand, P2 KFEKKFEK 3.6 mM peptides P3 and P4 showed very impressive inhibition at P3 RADSRADC 123 mM relatively low concentrations. The IC50 (half-maximal in- hibitory concentration) for P3 and P4 was 123 and 40 mM, P4 YRSRKYSSWY 40 mM respectively, compared with 680 mM for HQ. Figure 1 also P5 PLG 2.73 mM illustrates that the inhibitory activity of the peptides was dose- P6 VLLK No effect dependent, with P4 showing inhibitory activity at concentra- P7 KFEFKFEF No effect tions as low as 50 mM. On the basis of the above results, we 1 focused our attention on peptides P3 and P4. Figure 2 shows IC50 [M] is the concentration at 50% inhibition of mushroom tyrosinase in molar. the time–response curves and indicates that P3 and P4 exhibited similar inhibitory activity as HQ. Moreover, 1.0 1.0 P3 0.9 P4 0.8 P1 HQ P2 0.8 P5 OD (475 nm) 0.6 P6 P7 0.7 0.4 0.6 0.2 0.5 0.0 0.4 –4 –3 –2 –1 –5 –4 –3 –2 Log [M] Log [M] Figure 1. Dose–response curves for all seven peptides and HQ were drawn at 120 minutes post incubation with varying concentrations. The experiment was conducted in a 96-well flat-bottom plate. Each well contained 80 ml of 0.067 M potassium phosphate buffer (pH 6.8), 40 ml of 5 mg mlÀ1 L-tyrosine dissolved in 0.067 M potassium phosphate buffer (pH 6.8), 40 ml of the different concentrations of inhibitor in 5% DMSO solution, and 40 ml of 480 U mlÀ1 mushroom tyrosinase solution. The assay mixture was incubated at 37 1C and spectrophotometric readings at 475 nm were taken every 10 minutes. (a) P1, P2, P5, P6, and P7; (b) HQ, P3, and P4. 2 Journal of Investigative Dermatology
  3. 3. A Abu Ubeid et al. Oligopeptide Tyrosinase Inhibitors 1.4 0 150 1.2 0.01 0.02 125 0 1.0 0.05 0.8 100 0.05 0.3 1 / [Vo] 0.6 0.5 75 0.1 1 0.4 2 50 0.2 5 10 25 0.0 0 50 100 150 0 1.4 –60 – 40 –20 0 20 40 0 1/[S] 1.2 0.01 1.0 0.03 0.05 0.8 0.1 150 0 0.6 0.3 0.5 125 0.4 1 0.05 100 1/[Vo] 0.2 3 10 0.0 75 0.1 0 50 100 150 50 1.4 0.15 0 1.2 25 0.01 1.0 0.02 0 0.8 0.1 –60 – 40 –20 0 20 40 0.2 1/[S] 0.6 1 0.4 2 0.2 5 250 0.0 10 0 50 100 150 0 200 Figure 2. Time–response curves for peptides P3, P4, and HQ. The final 0.05 1/[Vo] volume of each well was 200 ml, containing 1 mg mlÀ1 L-tyrosine, 96 U mlÀ1 150 of mushroom tyrosinase, and varying concentrations of inhibitors, ranging from 10 mM to 10 mM. The 96-well plate was incubated at 37 1C and the 0.1 100 spectrophotometric readings taken every 10 minutes. Each point on the graph is an average of five independent trials, displayed with error 0.15 50 bars. (a) P3; (b) P4; and (c) HQ. 0 –60 – 40 –20 0 20 40 peptide P4 showed a more potent inhibitory effect at 1/[S] concentrations as low as 50 mM. Figure 3. Lineweaver– Burke plots. (a) P3 (b) P4 and (c) HQ. The concentration of mushroom tyrosinase [E] was 24 U mlÀ1. The series of Kinetics of mushroom tyrosinase inhibition by oligopeptides substrate concentrations [S] were 0.05 (or 0.07 for peptide inhibitors), 0.1, We next determined the kinetics of the inhibition for P3, P4, 0.15, and 0.2 mg mlÀ1. The series of inhibitor [I] were 0, 0.01, and 0.02 mM. and HQ using a standard assay (Pomerantz, 1966). The The results were analyzed according to the Lineweaver–Burke plot method inhibition mechanisms of the two peptides P3 and P4, as well that allows the determination of the Michaelis constant (Km) and maximum as that of HQ, are all competitive as shown by Line- velocity (Vm). All inhibitors showed a competitive inhibition pattern. weaver–Burke plots in Figure 3. The double reciprocal plots illustrate that Vmax was independent of substrate concentra- tion for all three inhibitors, whereas the Km was altered. The L-tyrosine as the substrate with L-dopa as the cofactor. In the inhibition constant (Ki) for HQ, P3, and P4 was determined to latter, L-dopa acts as a hydrogen donor helping eliminate be 0.053, 0.048, and 0.070 mM, respectively. the lag period observed during tyrosine hydroxylation (Pomerantz and Warner, 1967). Figure 4 shows dose- Inhibition of human tyrosinase by oligopeptides dependent inhibition for P3, P4, and HQ. It can be seen We also examined the inhibitory activity of P3 and P4 relative that P3 and P4 are more potent than HQ at concentrations as to HQ against human tyrosinase under two conditions: the low as 30 mM. At 100 mM, human tyrosinase activity was first using L-dopa as the substrate and the second using reduced by 35% for P4 and by 25% for P3. With L-dopa as the www.jidonline.org 3
  4. 4. A Abu Ubeid et al. Oligopeptide Tyrosinase Inhibitors L-Dopa L-Tyrosine 3 1.0 P3 P3 P4 P4 HQ 0.8 HQ 2 OD (475 nm) 0.6 0.4 1 0.2 0 0.0 –6 –5 –4 –3 –2 –6 –5 –4 –3 –2 Log [M] Log [M] Figure 4. Dose–response curves with human tyrosinase. Melanocytes were lysed using 1% Triton X-100 in 0.067 M potassium phosphate buffer (pH 6.8). Cells were then sonicated twice at 20% intensity for 30 seconds. Lysates were clarified by centrifugation at 10,000 g for 10 minutes after measuring protein content, an equal amount of 40 mg was added to each well and adjusted with lysis buffer to reach 150 ml. Each triplicate of wells received different concentrations (0–2 mM) of oligopeptide or HQ. (a) Using L-dopa as the substrate. L-Dopa was dissolved in lysis buffer and added to each well at a final concentration of 2.5 mM. The plate was incubated at 37 1C for 120 minutes, and spectrophotometric readings at 475 nm were taken at 10 minutes intervals. (b) Using L-tyrosine as the substrate with L-dopa at a final concentration of 0.005% as a cofactor. substrate, P3 was more potent than P4 at inhibiting the 45 enzyme. On the other hand, when L-tyrosine was used as the 40 substrate, P4 showed stronger inhibition than P3. These data support the hypothesis that tyrosinase has two distinct 35 Melanin content (pg per cell) catalytic sites for L-dopa and L-tyrosine (Olivares et al., 2001). 30 Effects of HQ, P3, and P4 on melanin content of melanocytes 25 To determine the effect of the oligopeptides on melanogen- 20 esis, we incubated melanocytes with HQ, P3, or P4 for 1 week and then measured melanin content. At a concentration 15 of 10 mM, HQ, P3, and P4 reduced melanin content by 7±2, 10 22±1, and 40±1%, respectively (Figure 5). At 100 mM, P3 and P4 reduced melanin content by 27±2 and 43±2%, 5 respectively, whereas HQ was found to be 100% toxic to 0 melanocytes. (µM) 1 10 100 1 10 100 1 10 100 Ctrl [HQ] [P3] [P4] Cytotoxic and proliferative effects of HQ, P3, and P4 on Figure 5. The effects of peptides and HQ on the melanin content of melanocytes melanocytes, expressed in pg per cell. The tested concentrations were 1, 10, To exclude the possibility that P3 and P4 were reducing and 100 mM. Melanocytes were incubated with test samples for a week. melanin content by inhibiting cell proliferation or inducing Cells were lysed and incubated O/N in 1 M NaOH. Melanin content was cytotoxicity, melanocytes were incubated in the presence or determined by comparing the absorbance results with a standard curve generated from synthetic melanin. absence of various concentrations of HQ, P3, or P4, and proliferation was assessed at various time points using the WST-1 Cell Proliferation Kit (Roche, Palo Alto, CA). Figure 6 shows that treatment with 100 mM or greater of HQ Table 2 further supports our findings regarding the toxicity completely abolished WST-1 activity (from 0.45±0.02 at of HQ on melanocytes compared to peptides P3 and P4. After day 1 down to 0.0 at day 5). These data are consistent with a 24-hour incubation of melanocytes with 10 mM HQ, our above findings and imply that HQ is cytotoxic to viability staining with trypan blue indicated that 27% of cells melanocytes at these concentrations. Unlike HQ, 10 mM, were dead. This was one order of magnitude greater than that 100 mM, or 1 mM P3 did not alter melanocyte proliferation for P4. The toxic effects of HQ were dose-dependent and at rates relative to the control group. Although no effect on concentrations of 30 and 100 mM, 41 and 98% of melanocytes melanocytes proliferation was observed for P4 at 10 and were no longer viable, respectively. The highest rate of cell 100 mM after 5 days of incubation, proliferation was inhibited death for P4 was 18% after incubating the melanocytes with by 10±2% at a concentration of 1 mM. 1 mM of P4 for 6 days. At concentrations 4100 mM for HQ, 4 Journal of Investigative Dermatology
  5. 5. A Abu Ubeid et al. Oligopeptide Tyrosinase Inhibitors Day 1 Table 2. The toxic effect of HQ, P3, and P4 5 % Cell death1 4 Day 1 Day 3 Day 6 OD (450 nm) Concentration [lM] P3 P4 HQ P3 P4 HQ P3 P4 HQ 3 1 0 2 10 0 3 12 0 3 10 2 10 0 3 27 0 6 29 0 5 48 30 0 3 41 0 4 53 0 5 76 1 100 0 4 98 0 4 100 0 7 100 0 300 0 6 100 0 11 100 1 9 100 1,000 0 10 100 0 15 100 2 18 100 Day 3 5 HQ, hydroquinone. 1 Human melanocytes were incubated with HQ, P3, or P4 at the above 4 concentrations for 1, 3, or 6 days. The mean percentage cell death for at least three independent runs is shown. OD (450 nm) 3 2 et al., 2003). The peptide dose- and time-response curves 1 showed that P3 and P4 were 6- and 17-fold more potent inhibitors of mushroom tyrosinase than HQ (IC50 values 0 presented in Table 1). On the basis of this initial screen, we decided to further evaluate and characterize the effects of P3 Day 5 and P4. 5 To determine the mode of inhibition, we determined Lineweaver–Burk plots of P3 and P4 and compared the 4 kinetics to those of HQ. These studies showed that similar to HQ, P3 and P4 were competitive inhibitors of tyrosinase. OD (450 nm) 3 Peptides P3 and P4 were also tested against fresh extracts of human tyrosinase. At a concentration of 100 mM, the peptides 2 were able to inhibit human tyrosinase activity by 25–35%. Although the potency against human tyrosinase appeared 1 reduced relative to that observed with mushroom tyrosinase, 0 several experimental conditions may account for this 10 1001,000 10 1001,000 10 1001,000 discrepancy. First, the assay was carried out on extracts from Ctrl HQ P3 P4 lysed melanocytes and not purified human tyrosinase. [µM] Although protease inhibitors were included to reduce degradation of human tyrosinase, we did not optimize Figure 6. Proliferation of human melanocytes treated with HQ, P3, and P4. these conditions. Second, the catalytic site of the extracted A total of 25,000 human melanocytes per well were incubated at 37 1C human tyrosinase can exist in different redox states than the in the presence of 10, 100, 1,000 mM HQ, P3, and P4 in Medium 254 for (a) 1, (b) 3, and (c) 5 days. After the incubation, 10 ml WST-1 was added purified form. to each well and absorbance was read at 450 nm after 4 hours incubation We observed a lengthy lag period for tyrosine oxidation. at 37 1C. The oxidation of L-tyrosine by tyrosinase has previously been characterized by a lag period that is affected by enzyme there was 100% cell death starting within 24 hours of and substrate concentration, pH, and hydrogen donors such exposure. P3 was found to be considerably less toxic to as L-dopa (Pomerantz and Warner, 1967). Therefore, when melanocytes even at concentrations as high as 1 mM (2% cell L-tyrosine was the substrate, L-dopa was added as a cofactor death after 6 days). at low micromolar levels to eliminate this lag period. The ability to act as substrate requires a free electron donor group, DISCUSSION whereas competitive inhibition, on the contrary, requires the In this study, we screened an internal library for oligopeptides presence of powerful electron acceptor groups (Passi and with inhibitory activity against mushroom and human Nazzaro-Porro, 1981). These experiments also unexpectedly tyrosinase. Even though the amino-acid sequence similarity showed that P3 inhibited human tyrosinase more potently in between mushroom and human tyrosinase is only 23%, our the presence of L-dopa than L-tyrosine, whereas P4 showed initial experiments were performed using mushroom tyrosi- greater inhibition in the presence of L-tyrosine as the nase as earlier studies suggested a strong correlation of substrate. Hearing and Ekel (1976) proposed that there are efficacy with agents tested against the human form (Sugimoto two catalytic sites on tyrosinase, one for tyrosine hydroxylase www.jidonline.org 5
  6. 6. A Abu Ubeid et al. Oligopeptide Tyrosinase Inhibitors and another site specific for dopa oxidase activity. Our toxicity, and minimal effects on melanocyte proliferation findings with P3 and P4 further support the hypothesis of rates. Further studies are under way to optimize cell separate catalytic sites for L-dopa and L-tyrosine (Schallreuter penetration and oligopeptide stability for transdermal et al., 2008). applications. We believe these oligopeptides may hold Despite the fact that little is known about the effect of significant therapeutic potential for the treatment of skin short-sequence peptides on melanogenesis and tyrosinase hyperpigmentation disorders. As both oligopeptides showed inhibition, there is great potential for these agents to replace substantially improved potency against tyrosinase with more toxic drugs such as HQ, as they comprise naturally minimal melanocyte toxicity, clinical use of these agents occurring amino acids that are easily metabolized in skin. may obviate the need for HQ, which derives its primary skin- Therefore, risks of systemic absorption and liver-related lightening capability through its cytotoxic activity. mutagenesis may be avoided during the chronic treatment of hyperpigmentation disorders. Some amino acids such as MATERIALS AND METHODS L-alanine, glycine, L-isoleucine, and L-leucine have previously Materials been shown to possess beneficial effects toward disruption of Mushroom tyrosinase, L-tyrosine, HQ, and L-dopa were purchased melanogenesis (Ishikawa et al., 2007). Ishikawa et al. from Sigma-Aldrich (St Louis, MO). The peptides (Table 1) were reported that a combination of these four amino acids had synthesized by NeoMPS (San Diego, CA) using solid-phase 9H- an additive effect on hypopigmentation similar to that of kojic (f)luoren-9-yl(m)eth(o)xy(c)arbonyl chemistry. acid. Schurink et al. (2007) proposed that the peptide should contain one or more arginine residues for strong tyrosinase Enzymatic assay of mushroom tyrosinase inhibitory and binding activity. Moreover, their studies Tyrosinase inhibition activity was determined in vitro using indicated that tyrosinase inhibition is optimal when arginine L-tyrosine as the substrate using a modified method from Piao and/or phenylalanine is/are combined with hydrophobic et al. (2002). The concentration of enzyme, substrate, and inhibitor aliphatic residues such as valine, alanine, or leucine. Besides was denoted as [E], [S], and [I], respectively. The experiment was hydrophobic residues, peptides containing the polar, un- conducted in a 96-well flat-bottom plate. Each well contained 80 ml charged residues cysteine and serine also showed significant of 0.067 M potassium phosphate buffer (pH 6.8), 40 ml of 5 mg mlÀ1 L- inhibitory activity. tyrosine dissolved in 0.067 M potassium phosphate buffer (pH 6.8), The amino acid tyrosine contains a phenol group, which 40 ml of the different concentrations of inhibitor in 5% DMSO makes it unique in that it can serve as a substrate or a solution, and 40 ml of 480 U per ml mushroom tyrosinase solution. competitive inhibitor. Oligopeptide P3 contains the combi- The final volume of each well was 200 ml, containing 1 mg mlÀ1 [S], nation Arg–Ala in positions 1/2 and 5/6, as well as the amino 96 U mlÀ1 [E], and varying concentrations of [I], ranging from 1 mM to acids serine and cysteine at positions 4 and 8, respectively. 10 mM. For the negative control wells, the inhibitors were substituted The presence of these key residues may explain the strong with 5% DMSO solution and adjusted to the total volume of 200 ml. inhibitory activity observed for P3. For P4, the presence of HQ served as the positive control in our experiments. HQ was tested tyrosine at positions 1 and 10, arginine at positions 2 and 4, in parallel with the oligopeptides at the same concentrations and and serine at 3, 7, and 8 may explain the robust inhibition same conditions. The assay mixture was incubated at 37 1C. The observed for this oligopeptide. optical density of the reaction mixtures, which correlates with the One of the main advantages of oligopeptides P3 and P4 is amount of dopachrome produced, was measured using a spectro- the relative absence of melanocyte cytotoxicity (Table 2). photometer (Varioskan, Thermo Electron Corporation, Waltham, Whereas HQ was 100% toxic after a 24-hour treatment at MA) at 475 nm and at different time periods. 100 mM, P3 showed no toxic effects after 6 days of treatment. P4 also showed minimal (7%; Table 2) toxicity after the same Kinetic studies incubation period. Treatment with 1 mM P3 resulted in a 2% The Lineweaver–Burk plot method was used to determine the toxicity rate (Table 2) and no effect on melanocyte prolifera- reaction kinetics (Burk et al., 1934). Experiments were conducted tion (Figure 6). At a concentration 25-fold higher than its IC50 using the same protocol used for measuring mushroom tyrosinase value, P4 began to show mild (18%; Table 2) melanocyte activity except for changes in concentrations of [E], [S], and [I]. [E] toxicity after a prolonged incubation of 6 days. We also was 24 U mlÀ1. The series of [S] were 0.05 (or 0.07 for peptide confirmed these findings using proliferation assays, which inhibitors), 0.1, 0.15, and 0.2 mg mlÀ1. The series of [I] were 0, 0.01, showed that HQ was toxic to melanocytes at concentrations and 0.02 mM. The results were analyzed according to the Line- as low as 30 mM (data now shown), whereas mild reduction of weaver–Burk plot method that facilitated the determination of the proliferation was first noted only after 3 days of incubation Michaelis constant (Km) and maximum velocity (Vmax). with 1 mM P4 (Figure 6). Lower concentrations did not show any effect on melanocyte proliferation even after prolonged Melanin content measurement incubation for 5 days, whereas HQ dramatically reduced Melanin content of the melanocytes was measured according to the melanocyte proliferation within 24 hours at concentrations as method of Naeyaert et al. (1991). Human melanocytes were cultured low as 30 (data not shown) and 100 mM (Figure 6). in 24-well plates and treated with individual test samples for 7 days. In conclusion, our findings suggest that oligopeptides P3 P3, P4, and HQ were tested at 10 and 100 mM each, all in triplicates. and P4 may be ideal tyrosinase inhibitors due to their potent Media were changed every other day and fresh oligopeptide or HQ enzymatic inhibitory activity, relative absence of melanocyte was added. After 7 days, the media were discarded and the wells 6 Journal of Investigative Dermatology
  7. 7. A Abu Ubeid et al. Oligopeptide Tyrosinase Inhibitors were washed with phosphate-buffered saline. Cells were detached 100, 300, or 1,000 mM). The plates were incubated at 37 1C in a by short incubation in trypsin/EDTA (0.25%/0.1% in phosphate- humidified 5% CO2 chamber. Media and inhibitors were changed buffered saline). An aliquot was used for cell counting and the every 48 hours. After 24 hours, the plate labeled ‘‘Day-1’’ was remaining cells were sonicated and incubated overnight in dark in harvested and the cells were counted in each well using a 500 ml of 1 M NaOH at 37 1C. Melanin concentrations were hemacytometer. The control well contained 2 ml of media without calculated by comparison of the optical density at 475 nm of any inhibitor. The toxicity of the test sample was measured unknown samples with a standard curve obtained with synthetic according to the following formula: melanin (Sigma-Aldrich). Melanin content was expressed as pg per ½1 À ðnumber of cells in control wellÞ À ðnumber of live cells in test sample wellފ cell. Â100% number of cells in control well The same procedure was repeated after 72 hours of incubation for the Assay of human tyrosinase activity plate labeled ‘‘Day-3’’ and after 144 hours for the ‘‘Day-6’’ plate. The following methods are modifications of protocols adopted from Nagata et al. (2004) and Cheng et al. (2007). Human melanocytes Statistical analysis were cultured in 100 mm dishes using Medium 254 (Invitrogen, Each experiment was performed in triplicates and run a minimum of Carlsbad, CA), incubated at 37 1C in a humidified 5% CO2 chamber. five independent times. The results were averaged, and standard At 80–90% confluence, cells were harvested and lysed using 1% error of the mean was calculated for all conditions using Microsoft Triton X-100 in 0.067 M potassium phosphate buffer (pH 6.8). Cells Excel. were then sonicated twice at 20% intensity for 30 seconds (on ice). Lysates were clarified by centrifugation at 10,000 g for 10 minutes. CONFLICT OF INTEREST Protein content was determined using a Bio-Rad protein assay kit The authors state no conflict of interest. (Richmond, CA). Lysates were added to a 96-well flat-bottom plate containing equal amounts of protein (40 mg), and adjusted with lysis buffer to reach 150 ml in each well. L-Dopa was dissolved in lysis REFERENCES buffer and added to each well at a final concentration of 2.5 mM. Briganti S, Camera E, Picardo M (2003) Chemical and instrumental Each triplicate of wells received different concentrations (0–2 mM) of approaches to treat hyperpigmentation. Pigment Cell Res 16:101–10 oligopeptide or HQ. 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Several control wells were used in the experiment. constituent from Podocarpus macrophyllus var. macrophyllus shows Certain wells served as controls and contained 40 mg of protein anti-tyrosinase effect and regulates tyrosinase-related proteins and mRNA in human epidermal melanocytes. Chem Pharm Bull (Tokyo) extract in lysis buffer, and substrate at varying compositions but 55:757–61 without peptide inhibitors. Other control wells contained lysis Curto EV, Kwong C, Hermersdorfer H, Glatt H, Santis C, Virador V et al. ¨ buffer, substrate, peptide inhibitors but no protein extract (no (1999) Inhibitors of mammalian melanocyte tyrosinase: in vitro compar- enzyme). isons of alkyl esters of gentisic acid with other putative inhibitors. Biochem Pharmacol 57:663–72 DeCaprio AP (1999) The toxicology of hydroquinone – relevance to WST assay for melanocyte proliferation occupational and environmental exposure. 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